Bluejuice loading buffer

10 pmol of purified sgRNA (IDT, Alt-R CRISPR-Cas9 sgRNA) alone, or 10 pmol of sgRNA and 10 pmol of purified spCas9 nuclease (Sigma-Aldrich, CAS9PROT-250UG), were incubated at room temperature for 10 min in a 20 μl reaction in 1 × 3.1 Buffer (B7203S, NEB) to form RNP. Specified dose of TAMRA dye labelled PNA was diluted into 1 μl of water, added to wells, and incubated in a thermocycler for 30 min at 37°C. A BIORAD 5% TBE polyacrylamide gel was pre-run for 30 min at 10 mA. 5 μl of final binding reactions were loaded onto gels with BlueJuice loading buffer (Invitrogen) and run at 10 mA. Gels were first imaged using BIORAD GelDoc XRS+ with a Green Epi (BIORAD #170-8284) 605/50 filter to acquire TAMRA signal and then incubated in 1× SYBR Gold (Invitrogen) in 1× TBE buffer for 5 min before imaging by standard UV transillumination (Raw images in Supplementary Figure S1). For band density analysis, three independent experiments were loaded onto a single 26-well gel and run according to the specifications above. Integrated densities for TAMRA-Cas9 co-localized bands were determined across wells using ImageJ analyses, and background signal from each no PNA (0 pmol) control was subtracted for each experiment.